Smooth muscle (SM) forms the wall of all hollow organs including blood vessels and the airway. Misregulation of SM contractility leads to hypertension, asthma, cerebral vasospasm, urinary incontinence, erectile dysfunction and others. Importantly, in each of these diseases a different SM tissue type is affected and constitutes a critical target for drug design to selectively target for example small blood vessels for treatment of hypertension or the airway for asthma. The small GTPase RhoA is an important player regulating contraction through inhibition of myosin phosphatase. RhoA is controlled by many different GTP exchange factors (GEFs) and GTPase activating proteins (GAPs) and these unique auxillary regulatory pathways constitute potential targets for tissue selective therapies. We identified p190RhoGAP, GEF-H1, ArhGEF17 and GrinchGEF as unexplored potential key players and will define their molecular mechanisms of action and how they modulate RhoA-mediated SM contractility. Relevant protein constructs and functional assays are in place for all Aims. We bring our synergistic approach bridging our expertise in physiology (A.V. Somlyo) and structure-function relationships (Z.S. Derewenda) to solve the role in SM of these multidomain GEFs and GAPs. Aim 1 To define how p190RhoGAP modulates RhoA-mediated SM contractility. We suggest, that up and down regulation of p190RhoGAP activity is important for sustaining RhoA mediated Ca2+-sensitized contraction and relaxation. We will dissect the p190 signaling pathways and its putative regulatory partners c-Abl, SHP2 and Rnd3. We will assess how supramodular rearrangements regulate autoinhibition and activation in p190RhoGAP and assay the roles of these recombinant domains in RhoA mediated contractions. Aim 2 To determine the physiological functions and molecular mechanism of GEF-H1. We will silence GEF-H1 and investigate whether response to increased intraluminal pressure (myogenic response) or the agonist- RhoA-mediated contractile response is lost. Structure-function relationships will be determined using a synergistic combination of crystallography, SAXS, NMR and biochemistry to investigate the catalytic and regulatory mechanisms of GEF-H1. We expect that GEF-H1 couples through distinct GPCRs or responds to increased intraluminal pressure to increase RhoA activity contributing to different SM contractile phenotypes. Aim 3 To characterize the physiological role and structure-function relationships in the ArhGEF17 family. ArhGEF17 is a newly discovered RhoA-selective GEF with a unique domain structure, including a split-PH domain and a WD40 domain. These GEFs are expressed but their function in SM is unknown. The significance of our research is in identification and exploration of new or inadequately studied signaling pathways in SM tissues, moving forward the field of SM physiology and structural biology, while opening opportunities for innovative drug discoveries.